Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery (original) (raw)
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Biomedit͡sinskai͡a khimii͡a
The electrochemical reduction of the recombinant form of human cytochrome P450 17A1 (CYP17A1) was investigated. Hemeprotein was immobilized on electrode modified with biocompatable nanocomposite material based on the membrane-like synthetic surfactant didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of DDAB/Au/CYP17A1 electrodes were investigated with cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Analysis of electrochemical behaviour of cytochrome P450 17A1 was conducted in the presence of substrate pregnenolone (1), inhibitor ketoconazole (2), and in the presence of synthetic derivatives of pregnenolone: acetylpregnenolone (3), cyclopregnenolone (4), and tetrabrompregnenolone (5). Ketoconazole, azole inhibitor of cytochromes P450, blocked catalytic current in the presence of substrate pregnenolone (1). Compounds 3-5 did not demonstrate substrate properties towards electrode/CYP17A1 system. Compound 3 did ...
Analytical Chemistry, 2010
Oriented immobilization of human cytochrome P450 2E1 and its catalytic activity by direct electrochemistry was achieved by engineering two multisite mutants of P450 2E1: MUT261 (C268S-C480S-C488S) and MUT268 (C261S-C480S-C488S). Here, all the exposed cysteines are mutated into serines, with the exception of one (C261 for MUT261 and C268 for MUT268) that is able to link covalently to a modified gold electrode. The P450 2E1 wild type, as well as the two mutants, were immobilized onto gold electrodes using dithio-bismaleimidoethane as a self-assembled monolayer. The catalytic activity of the wild type and of the two cysteine mutants were determined using p-nitrophenol as a substrate, and the amount of the electrocatalysis product (p-nitrocatechol) was determined spectrophotometrically. The amounts of product formed by the mutants on the electrodes were 2-fold to 3-fold higher than those of the wild type. Control experiments performed in solution using the cytochrome P450 reductase as the electron donor show no significant differences in the level of product formed. The higher level of product formation of the two mutants on the electrode is ascribed to the controlled immobilization on the gold surface: the heme electron transfer proximal side is linked to the electrode, while the substrate binding distal side is exposed to the bulk solution. This is the first evidence that the control over the orientation of the human cytochromes P450 is key to maximize the electrocatalytic efficiency of these enzymes.
Preparation, Characterization, and Substrate Metabolism of Gold-Immobilized Cytochrome P450 2C9
Journal of the American Chemical Society, 2006
Procedures and key data for the 1) preparation of CYP2C9-gold substrate, 2) AFM, SQUID magnetometry, and UV measurements, 3) CYP2C9-gold substrate metabolism of THC, and 4) CYP2C9 molecular modeling are presented here. General. Methanol, ethanol, glacial acetic acid, ammonium acetate, and THAM were obtained from Fisher Scientific (Pgh, PA). Δ 9-THC, 11-hydroxy-Δ 9-THC, 2 H 3-Δ 9-THC (both 5,5,5trideuteropentyl compounds) were purchased from Cerilliant (Round Rock, TX, USA). All other chemicals and solvents were obtained from Aldrich (Milw., WI) and used as received unless otherwise noted. All surface modification reactions were conducted under dry argon. The CYP2C9 used was prepared by expression in an E. Coli system, isolated, and purified as previously described. 1 Cytochrome P450 reductase (human) was purchased from Invitrogen (Carlsbad, CA). Enzyme-gold substrate preparation: Gold substrate coated silicon wafers were prepared by first depositing 5.0 nm of tantalum (for improved adhesion) and then 5.0 nm of gold via dc sputtering in a 3 mTorr atmosphere of Ar. Prior to enzyme attachment, the gold surface was cleaned by soaking in piranha solution for 30 min, washing with water, drying under argon and then repeating this procedure with hexane, ethanol and finally nanopure water. One of two different linkers (A or B) was then attached as described below. Linker A: Gold substrates were treated with cystamine hydrochloride in water (0.1 M) for 24 h S2 at rm temp. The gold substrates were then washed with water, ethanol, and dried. The gold substrates were then covered with a solution of 3-maleimidopropionic acid Nhydroxysuccinimide ester in DMF (0.1 M) and allowed to stand for 24 h. 2 The gold substrates were again washed with water, absolute ethanol, and dried. Finally, the gold substrates were immersed in a solution of CYP2C9 (3.3 μM, 200 μL, 0.6 nmol) in sodium phosphate buffer (1 mL, 40 mM, pH 7.4) and allowed to stand for 24 hours at rm temp. Samples containing flurbiprofen, dapsone, or flurbiprofen and dapsone, in addition to CYP2C9, were prepared in the same way except that the CYP2C9 solution also contained flurbiprofen (1 mL, 40 μM) or flurbiprofen and dapsone (1 mL, 40 μM each). Linker B: Gold coated substrates were treated by immersing them in a solution of 8mercaptoundecanoic acid (MUA) (25 mM) and 1-octanethiol (OT) (75 mM) in ethanol, at rm temp, for 24 h. 3 The gold substrates were then washed with absolute ethanol, dried, and treated with a solution containing N-((3-dimethylamino)-propyl)-N-ethyl carbodiimide hydrochloride (EDC) (1.0 mM) and N-hydroxysulfosuccinimide (NHS) (2.5 mM) in phosphate buffer (40 mM, pH 7.4), at rm temp, for 1 h. 4 The gold substrates where then washed with phosphate buffer (40 mM, pH 7.4), and immersed in a solution of CYP2C9 alone, CYP2C9 and flurbiprofen, CYP2C9 and dapsone, or CYP2C9, flurbiprofen, and dapsone as describe under Method A at rm temp, for 24 h. AFM. The AFM system used was a Multimode scanning probe microscope (SPM) run by Nanoscope IIIA electronics (Veeco Instruments, Woodbury, NY). AFM images were obtained by first mounting the gold-enzyme samples on a magnetic AFM sample disk and placing the disk
Biosensors and Bioelectronics, 2005
This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) and gold nanoparticles immobilised on rhodium-graphite electrodes. Thin films of gold nanoparticles were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto both gold nanoparticle modified and bare rhodium-graphite electrodes. Cyclic voltammetry indicated enhanced activity of the enzyme at the gold nanoparticle modified surface. The role of the nanoparticles in mediating electron transfer to the cytochrome P450scc was verified using ac impedance spectroscopy. Equivalent circuit analysis of the impedance spectra was performed and the values of the individual components estimated. On addition of aliquots of cholesterol to the electrolyte bioelectrocatalytic reduction currents were obtained. The sensitivity of the nanoparticle modified biosensor to cholesterol was 0.13 A M −1 in a detection range between 10 and 70 M of cholesterol. This confirms that gold nanoparticles enhance electron transfer to the P450scc when present on the rhodium-graphite electrodes.
Scientific reports, 2016
Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, "nanodiscs", and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and -300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(-1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2)...
Inhibition of Human Cytochrome P450 Enzymes by Metallic Nanoparticles: A Preliminary to Nanogenomics
International Journal of Pharmacology, 2008
Objective: 1,4-Dihydropyridine calcium antagonists such as nifedipine are potent vasodilators. It is now commonly agreed that the oxidation of 1,4dihydropyridine into pyridine, which is one of the main metabolic pathways, is catalysed by the cytochrome P450 (CYP) 3A4 isoform. In the present study, the inhibitory eects of 13 kinds of 1,4-dihydropyridine calcium antagonists clinically used in Japan on human CYP-isoform-dependent reactions were investigated to predict the drug interactions using microsomes from human B-lymphoblast cells expressing CYP. Results: The speci®c activities for human CYP isoforms included 7-ethoxyresor®n O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), 7-benzyloxyresoru®n O-dealkylation (CYP2B6), S-warfarin 7-hydroxylation (CYP2C9), S-mephenytoin 4¢-hydroxylaion (CYP2C19), bufuralol 1¢-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and testosterone 6b-hydroxylation (CYP3A4). Benidipine and amlodipine competitively inhibited the CYP1A1 activity. Nifedipine, nisoldipine and aranidipine competitively inhibited the CYP1A2 activity. No 1,4-dihydropyridie calcium antagonists used in this study inhibited the CYP2A6 activity. Barnidipine and amlodipine inhibited the CYP2B6 activity. Nicardipine, benidipine, manidipine and barnidipine competitively inhibited the CYP2C9 and CYP2D6 activities. Inhibition extent of the CYP2E1 activity by nifedipine and aranidipine were weak. Nicardipine, benidipine and barnidipine inhibited the CYP2C19 and CYP3A4 activities. Among the human CYP isoforms investigated, the inhibitory eects of 1,4dihydropyridine calcium antagonists were potent on human CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6 as well as CYP3A4. Furthermore, the isoform selectivity of inhibition by 1,4-dihydropyridine calcium antagonists was clari®ed. Conclusions: In consideration of the K i values obtained in the in vitro inhibition study and the concentration of 1,4-dihydropyridine calcium antagonists in human liver, the possibility of in vivo drug interactions of nicardipine and other drugs which are mainly metabolised by CYP2C9 and/or CYP3A4 was suggested. The inhibition of human CYP isoforms by 1,4-dihydropyridine calcium antagonists except nicardipine might be clinically insig-ni®cant.
A novel method for direct electrochemistry of a thermoacidophilic cytochrome P450
2006
Direct electron transfer of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7, (P450st) entrapped onto the surface of a plastic formed carbon electrode in poly(ethylene oxide) (PEO) containing 0.5 M KCl was achieved. The formal potential for wild-type P450st is À103 mV (vs. Ag wire). Thermophilic P450st was modified with PEO having an average molecular weight of 2000 (PEO 2000) to increase its stability in PEO solvent. The P450st enzyme was dissolved in several organic media and PEO oligomers after the modification. Resonance Raman spectra revealed no effects of chemical modification with PEO 2000 and PEO solvent on the heme structure of P450st. The electrochemical responses of PEO 2000-modified P450st in PEO solvent were well-defined and stable compared with those of wild-type P450st. Voltammetry of the modified protein on the surface of a plastic formed carbon electrode in PEO solvent showed a Fe III /Fe II potential at À114 mV vs. Ag wire, which is similar to that of wildtype P450st.
Electrochemical investigations of fungal cytochrome P450
Journal of Electroanalytical Chemistry, 2011
The electrochemical investigations of fungal (Aspergillus terreus MTCC 6324) cytochrome P450 monooxygenase (CYP) (EC 1.14.14.1) immobilized on multi-walled carbon nanotubes–Nafion®–polyethyleneimine (MWCNT-NF/PEI) modified glassy carbon electrode (GCE) were carried out to understand the electrochemistry of the CYP and its application potential in electrochemical-based devices. In deoxygenated condition, the formal potential for Fe(III)/Fe(II) redox couple of CYP was about −0.53 V (vs. Ag/AgCl reference electrode, pH 8). A positive shift in the redox potential of the bioelectrode to −0.475 V was observed in the presence of oxygen and substrate. The electrocatalytic activity towards n-hexadecane and inhibition with piperonyl butoxide (PBO) confirmed the involvement of Fe(III/II) system of CYP in the redox process. The current response increased linearly from 1 μM to 100 μM of n-hexadecane with detection limit 0.1 μM. The IC50 for PBO was 2.7 μM. The surface coverage of CYP immobilized...